ENZYMATIC METHOD FOR SYNTHESIS OF O-GLYCOSYLATED AMINO ACID OR PEPTIDE OR DERIVATIVES THEREOF

Abstract: Present invention describes a method for synthesis of O-glycosylated amino acid or peptide or derivatives thereof, characterized by the use of a glycosyl donor which is a monosaccharide in pyranose or furanose form or which is an oligosaccharide, and by the use of an acceptor which is a hydroxyl group containing amino acid or peptide derivative which has been modified in its N-terminal alpha -amino group and in which the C-terminal carboxyl group is non-modified, employing an exo- or an endoglycosidase (EC 3.2) as catalyst in an equilibrium reaction and that the product is isolated from the reaction mixture or is used directly or after partial or complete isolation in a continued synthesis with chemical methods or with enzymatic methods.

Description: Enzymatic method for synthesis of O-glycosylated amino acid or peptide or derivatives thereof. The present invention describes a new method to produce O- glycosylated amino acids, O-glycosylated peptides or derivatives of these. In a further aspect the present invention relates to products produced by the above method as well as uses of the resulting products. Glycoconjugates contain oligosaccharide chains with up to twenty monosaccharide units and several sequences have been shown to have biological activity e.g. in the binding to different cells, pathogens, toxins, antibodies or other proteins on cell surfaces, in cancer metastasis, in inflammation processes (for example selectin-carbohydrate interactions) , as a modifier of the activity, stability and biological activity of proteins, and as immunogenic substances which have potential for vaccination against different diseases. An extensive literature has been developed during the last few years in this field and there are several review articles on this type of biology, glycobiology, e.g. in Annual Review of Biochemistry and in Current Opinion in Structural Biology (see for example volume 3, 1993) incorporated herein by reference. One type of glycoconjugate, the glycoproteins, contain carbohydrate-peptide sequences in which the carbohydrate unit is bound to the peptide or protein chain via mainly three different types of linkages, the O-glycosidic linkage represented by GalNAcα-OSer and GalNAcα-OThr, that is the linkage between N- acetyl-D-galactosamine and the hydroxyl group on a L-serine or a L-threonine residue in the peptide or protein chain (several such linkages can be found in a peptide or protein chain depending on the number of serine or threonine units in the molecule) , the N-glycosidic linkage between N-acetyl-D- glucosamine and the amide function in asparagine, GlcNAc/3-N-Asn, and the O-glycosidic linkage between galactose or xylose and different hydroxyl group containing amino acids. Later, also the β-O-glycosidic linkage between N-acetyl-D-glucosamine and the hydroxyl group on serine or threonine, abbreviated GlcNAcβ- OSer and GlcNAc -OThr, respectively, has been of increased interest recently, e.g. because of its suggested importance in the DNA-replication in the nucleus of eucaryotic cells. These types of linkages are of interest to produce by synthetic methods for fundamental studies and for synthesis of biologically or pharmaceutically active fragments of glycoproteins, for instance for use as vaccines or therapeutics. It is also important to be able to synthesize analogs or derivatives of the structures above for example with other types of sugar and/or configuration, as e.g. mannosamine and the - or β- configuration to modify or to increase the biological activity of the conjugate. Background literature to organic synthesis of glycosidic linkages (Crout, D. , et al., (1991), J. Chem. Soc, Chem. Commun. 1550-1551; Nilsson, K.G.I. (1987), Carbohydrate Res. 167: 95-103; Nilsson, K.G.I. (1988), Trends Biotechnol. 6: 256- 264; Nilsson, K.G.I. (1991), ACS Symposium Series 466, 51-62) and to glycoconjugates in general (Feizl, T. (1993) , Curr. Opin. Struct. Biol. 3: 701-710; Karlsson, K.-A. (1989), Ann. Rev. Biochem. 58: 309-350). The carbohydrate part in glycoproteins is bound to the protein via a glycosidic N-linkage or via a glycosidic O-linkage, e.g. a galactosyl-serine or a galactosyl- threonine linkage (Parekh, R.B., (1991), Curr. Opin. Struct. Biol. 1: 750-754). Recently, efforts to prepare these linkages via either multi-step chemical procedures (Meldal and Bock, 1990) , or via stereospecific enzymatic procedures (Johansson, E. , et al. (1991), Enzyme Microb. Technol. 13: 781-787; Cantacuzene, D. and Attal, S. (1991), Carbohydrate Res. 211: 327-331) have been reported. Glycosidases have been utilized in a few cases. Thus, it was found that glycosylated products were obtained under equilibrium conditions employing monosaccharide and non-modified serine or threonine in high concentrations (Johansson et al 1991) . However, in this type of reaction, the N-glycosylation of the reducing end of the monosaccharide is a severe chemical side-reaction. Interestingly, enzymatic glycosylation of serine or threonine was not observed under kinetic (transglycosylation) reaction conditions, i.e. employing disaccharides or activated glycosides as glycosyl donors. However, it was later found that if serine or threonine was reacted (or modified) with protection groups in both the amino and carboxyl functions, the resulting protected amino acid derivative could be used as an acceptor in a /3-galactosidase-catalyzed kinetic reaction employing lactose or nitrophenyl /3-galactoside as the glycosyl donor (Cantacuzene, D. and Attal, S. (1991), Carbohydrate Res. 211: 327-331). The disadvantages with chemical synthesis is that several reaction steps are required with extensive protection group chemistry to obtain stereo- and regioselective synthesis of the conjugate and often, as a side reaction, jS-elimination and racemization of the amino acid residue is obtained due to the weakly acidic nature of the optically active C-H linkage. Enzymatic synthesis can be carried out with glycosyltransferases (EC 2.4) and glycosidases (EC 3.2). Glycosidases have been used in either reversed hydrolysis (equilibrium) reactions or transglycosylation (kinetic) reactions, or in general (see also K. Nilsson, Trends in Biotechnology, 1988, 256, incorporated herein) : Sugar-OR + HO-Aminoacid (or HO-Peptide/Derivate) H 2 0 a- or jS-glycosidase Sugar-OH + ROH Sugar-O-Aminoacid (or HO-Peptide/Derivate) + ROH where R represents H in an equilibrium reaction, or represents an organic group, i.e. a saccharide unit or an aromatic or aliphatic group in a kinetic reaction. Simple monosaccharides have been used as donor, Sugar-OR, in equilibrium synthesis, whereas various O-glycosides thereof including lactose, have been used in transglycosylation reactions. The enzyme is chosen with regard to the sugar substrate and linkage to be prepared (with α-N-acetyl- galactosaminidase one obtains GalNAcα-Ser/Thr, as is known in the art) . The disadvantages with earlier enzymatic techniques are several: In the kinetic reaction synthesis have been achieved only by using serine and threonine derivatives protected in both the o.-amino (N-protected) and carboxyl groups (C-protected) and then only with galactosides as donors, and in equilibrium controlled reactions side reactions such as N-glycosylation has been obtained, because unprotected amino acids as acceptor have been used. Another disadvantage with kinetic reactions is that the galactosides which have been used to achieve acceptable yields, nitrophenyl galactosides, often are relatively expensive and may be more or less difficult to separate from the product. According to the method of the present invention the synthesis of glycosylated amino acid or peptides or derivatives thereof is produced by the reaction of (a) a simple sugar in pyranose or furanose form as glycosyl donor, for example N- acetyl-glucosamine (GlcNAc) , N-acetyl-galactosamine (GalNAc) , mannose (Man) , xylose (Xyl) , galactose (Gal) , fucose (Fuc) , or specific oligosaccharide sequences which contain one or more of any of these monosaccharides, and (b) hydroxyl group containing amino acid or peptide derivative as acceptor which contains at least one hydroxyl group and which has been modified in its (N-) terminal α-amino function but which has a non-modified C- terminal carboxyl group, employing an exo- or endoglycosidase as catalyst (enzyme) . This enables, according to the invention, synthesis of glycopeptide fragments as well as derivatives thereof the type X-GalNAcα-OR, X-GalNAc/3-OR, XGalcx-OR, XGal3-OR, XManα-OR, XManβ- OR, X-Glcα-OR, X-Glc/3-OR, X-Xylα-OR, X-Xylβ-OR, X-GlcNAcα-OR, X- GlcNAc/3-OR, X-Fucct-OR, wherein X is a glycosidically bound carbohydrate-group (by using endoglycosidase) , or, when an exoglycosidase is used, X is not present (i.e. X-GalNAcα-OR, etc is the same as GalNAcα-OR, etc.). The acceptor, HOR, consists of a hydroxyl group containing amino acid or peptide which has been modified in at least the terminal N-group, but which has a free (non-modified) carboxyl group. Examples of such acceptor substances are R'-serine, R'-threonine, R'-Ser-Ala-OH, R'-Ser-Val- OH and so on, where R 1 is a protection group on the amino function of the amino acid or peptide such as an acetyl (Ac-) , t-butyloxycarbonyl (Boc-) or Fmoc-group. Thus, in general when a serine derivative is used as acceptor, the acceptor used according to the invention can be illustrated as follows: NHR 1 HOCH 2 C(H)C(0)R 2 where R 1 is a protection group on the amino group, or in the case when the acceptor is a peptide R 1 represents an amino protection group or an amino acid- or peptide group which is protected in the α-N-terminal group with an amino protection group, and where R 2 consists of an -OH group, or represents an amino acid or peptide group (bound via a C(0)-NH linkage) which has a non-modified carboxyl group in the carboxyl terminal unit. The amino protection group is described above and below. Preferably, according to the method of the invention, the donor is used in high concentration (preferably higher than IM when buffered water is used as the only solvent) or in excess over the acceptor, since the donor (simple monosaccharides with exoglycosidases) is abundant and have high solubility in water. In order to speed up the reactions and/or to achieve high substrate concentrations, relatively high concentrations of enzyme (e.g., 1-50 U/ml where 1 U is defined as the hydrolysis of 1 μmole of glycoside substrate per minute) and often increased temperature (room temperature or higher, e.g. 25°— 65°C) may be used. However, in some cases yields may be improved if lower temperatures are used, such as when organic solvents are used. The glycosidase can be used preferably in a relative pure form to obtain a stereospecific synthesis. Among traditional N-protection groups (R 1 above) which may be used according to the invention may be mentioned alkoxy and aliphatic protection groups e.g. benzyloxycarbonyl-, allyloxycarbonyl-, t-butyloxycarbonyl- (Boc-) , formyl-, acetyl-, fenacetyl-, 4-metoxybenzyloxycarbonyl- (Moz-) , 9-fluorenyl methyloxycarbonyl (Fmoc-) , etc. (see for example Houben-Weyl, Bd 15/1, Kontakte 3/79, 14 and especially Synthetic Peptides, G.A. Grant, Editor, W.H. Freeman and Company, New York, 1992, both of which are incorporated by reference in their entirety) . After the synthesis according to the invention one can, if desired, remove the N-protection group with standard chemical techniques (see Grant supra) . If a peptide derivate is acceptor in the reaction according to the invention, the size is preferably di- to penta peptide but even bigger peptide fragments can be glycosylated according to the invention. With the method according to the invention glycosylation can be achieved also of hydrophobic amino acid or peptide derivatives because of the high concentration of donor and the equilibrium type of reaction facilitates glycosylation of acceptors which are inefficient acceptors in kinetic reactions (which require an efficient acceptor and a relatively high concentration of acceptor to minimize the hydrolytic side reaction) . Also, acceptors used in the method according to the invention have a free carboxyl group which facilitate their dissolution in solvents with high water content and thus no or low amounts of cosolvents are required. However, the reactions according to the invention may also be carried out in mixed solvents, i.e. of water and water-miscible organic solvent, or of water and water-immiscible solvent or of two phase systems made up of water and polymer(s). The enzyme may be used in immobilized, cross-linked or in soluble form. The enzyme may be used adsorbed to a solid phase such as e.g. glass or polystyrene. Examples are the use of enzyme adsorbed to e.g. celite or XAD R resins. The latter may be especially useful in the cases when organic solvents (acetone, acetonitrile, tetrahydrofurane) are used as cosolvents (in high concentrations e.g. > 70%) or in two-phase systems. Moveover, and an important advantage of the method according to the invention, is that the product can be isolated by for example (a) slight acidification of the reaction mixture (pH 4-5; neutralizing the charge on the carboxyl group) followed by (b) precipitation or extraction of unreacted acceptor with a suitable organic solvent, followed by for example (c) column chromatography (e.g. on a Sephedex R , silica, reversed phase or ion-exchange column) or another extraction step with a less hydrophobic solvent to remove the product from the reaction mixture (the unmodified sugar will stay in the water phase) , or followed by evaporation of the water phase and extraction with for example methanol eventually followed by column chromatography of the methanol phase. Below a few examples, which in no way are meant to limit the scope of the invention, are given. EXAMPLES The choice of suitable conditions, as determined by pH (usually in the range 4-8 depending on enzyme, type of donor and solubility of acceptor) , reaction time (usually the reaction is followed by thin layer chromatography, HPLC, NMR, GC or another suitable method and stopped at or close to equilibrium) , temperature, solvent composition (often with small amounts, below 50% V/V, or in several cases preferably without organic cosolvent to speed up the reaction, see above) , do not limit the invention and are chosen with regard to the specific reaction. Mannose was dissolved in water (>40% w/w) together with Boc-L-serine (in this example 0.6 M) α-Mannosidase was added with pH corrected to 6.5. The reaction was carried out at 40° Celsius and after a suitable reaction time, depending on the amount of enzyme activity added and reaction temperature, the product (N-t-Boc)-L-serine α-mannoside (compound III in the Figure below) , was isolated by (a) extraction of the water phase to remove the excess of the acceptor followed by column chromatography of the water-phase. The donor can be used in extremely high concentrations (up to ca 85% w/w or higher) and at higher temperatures than mentioned above. The acceptor can also be used in high concentrations, often higher than 0.5 M, because the non-modified deprotonated carboxyl group increases the water solubility of the acceptor. A typical equilibrium controlled synthesis of Manα-Ser(N- Boc) (compound III in the figure below) was carried out by dissolving Boc-L-serine (compound I in the figure below) (700 mg) in buffer (0.05 M) , the pH was adjusted to 5.0, the liquid volume adjusted to 1.6 ml followed by the addition of mannose (2.5 g) . After the addition of α-mannosidase (20 U) the reaction was carried out at 25°C for five days. The reaction was terminated by heat treatment in a boiling water bath for 3 minutes and the excess of Boc-L-serine was removed by extraction of the water phase with methylene chloride. The product was isolated by column chromatography " (Kiselgel; 40-60 μm; Merck, Darmstadt) . The fractions containing the product were identified with TLC (eluent as above) , evaporated, rechromatographed on silica and the product was kept in dry form until analyzed with NMR. NHBoc NHBoc H0-CH,-C-H H0-CH(CHι 3 ,)l-C-H Boc: -C-0-C(CH 3 ) 3 COOH COOH II III IV V VI A similar type of synthesis as above with (N-t-Boc-L-serine as acceptor and with galactose or GalNac as the glycosyl donor and /3-galactosidase or α-galactosaminidase, respectively, as the catalyst were carried out to give (N-t-Boc)-serine β-O- galactoside (V) and (N-t-Boc)-serine N-acetyl-α-D- galactosaminide (VII) as illustrated below: /3-galactosidase -0-C(CH 3,)'3 α-N-Acetyl-D-Ga lactosami nidaεe VII A typical equilibrium controlled synthesis of (N-t- Boc) -L-serine β-galactoside (compound V below) Galβ-Ser(N-Boc) was carried out at pH 5 in a total liquid volume of 2 ml following the above procedure for dissolving Boc-L-serine (900 mg) and galactose (2.1 g) . After the addition of β- galactosidase (100 U) , the reaction was carried out at 35°C for ten days. Termination of the reaction and isolation of product was performed as above. A similar type of synthesis as above with (N-t-Boc) -L- serine as acceptor and with glucosamine as glycosyl donor and with jS-glucosaminidase as the catalyst was carried out to give (N-t-Boc)-serine N-acetyl-/3-D-glucosaminide. A similar type of synthesis as above with (N-t-Boc) -L- serine as acceptor and with N-acetyl-1) -galactosamine as glycosyl donor and with -galactosaminidase as the catalyst was carried out to give (N-t-Boc)-serine N-acetyl-β-D- galactosaminide. A similar type of synthesis as above with (N-t-Boc) -L- serine as acceptor and with for example glucose, xylose, as glycosyl donor and with a suitable α-glucosidase, β-glucosidase, α-xylosidase, β-xylosidase, respectively, as catalyst, may be carried out to produce the corresponding (N-t-Boc) -L-serine α- or jS-glycosides. The same type of reaction as above with Boc-D- serine or the corresponding threonine derivatives (see compounds II, IV and VI supra) may be carried out. By the use of other N-blocked derivatives of the type (N- X)Y- (as exemplified in the formula supra) , where X is an N α - amino protection group, such as for example Boc-, or Fmoc- or another α-N-protection group which has been exemplified above, and where Y can be-serine or threonine or a peptide residue containing any of those amino acids according to the formula above, then the above mentioned types of donors and enzymes can be used in a corresponding manner to produce the corresponding glycosylated derivative of the respective type of acceptor. Amino acid or peptide derivatives of the D-configuration or which contain a mixture of amino acid residues of the D-and the L-configurations may be used as acceptors according to the invention. Glycosidases are abundant in all living organisms and the source of enzyme do not limit the scope of the invention. Examples of suitable sources are different microorganisms such as bacteria and yeast (e.g. Aspergillus niger and Asperg±llus oryzae) , plants and different animal tissues. Depending on the desired product the enzyme can be used in more or less isolated form, if a stereospecific reaction is required the enzyme should be free of any contaminating glycosidase which may lead to formation of the non-desired anomeric configuration. The enzyme may be used in soluble or immobilized form (to allow reuse if desired) as bound covalently or adsorbed to a solid support (e.g. a glass, silica, polystyrene, another plastic, a polysaccharide (e.g. cellulose or agarose) ) or enriched in a water phase in a two-phase system. The product can be used directly, or after the removal of the N-protection groups, e.g. by chemical or enzymatic methods such as treatment with lipase or protease, or after the binding to other molecules via e.g. the amino group or the carboxyl group, e.g. attachment to polymers, plastics, metal surfaces, including gold surfaces via e.g. a mercaptopropionic acid spacer, ELISA-plates, proteins, lipids (the carboxyl or amino group can be used for covalent binding) in biological studies or can be used in a continued chemical or enzymatic synthesis according to the invention. The product can for example also be used for synthesis of disaccharide amino acid or peptide derivatives. Consequently, it is possible with the method according to the invention to (a) synthesize one of the glycosylated derivatives as mentioned above and (b) use such a glycosylated derivative as acceptor for synthesis with glycosyltransferase or glycosidase of for example the following type of structures (the (N-X)Y structure has been defined above; this group may have been modified as described above before the synthesis described below) : Gal / Sl-3GlcNAc / S-(N-X)Y or Gal/3l-3GalNAcα-(N-X)Y by using β- D-galactosidase (for instance from ox testes) as catalyst, nitrophenyl β-D-galactoside or lactose as donor and GlcNAc/?-(N- X)Y or GalNAcα-(N-X)Y as acceptor, respectively, Gal/51-4GlcNAcjS-(N-X)Y by using a suitable -galactosidase as catalyst, nitrophenyl β-D-galactoside or lactose as donor and GlcNAc/3-(N-X)Y as acceptor, Manαl-2Manα-(N-X)Y by using α-D-mannosidase as catalyst, nitrophenyl α-D-mannopyranoside as donor and Manα-(N-X)Y as acceptor. In addition to the above a number of other oligosaccharide sequences can be produced with other glycosidases (e.g. by the use of glycosidases mentioned above) and/or other acceptors. Glycosylation with mannose as the glycosyl donor is attractive for the synthesis of O-mannosylated serine or threonine derivatives due to the abundant donor substrate and the stereospecific reaction. As expected, the formation of product was more rapid at the higher temperatures (in the range 25°-50°C) . Moreover, the yield of product increased with the reaction temperature in the range 25°-50°C. The influence of using a higher substrate concentration (7 M mannose and 2 M Boc-serine) was investigated. The initial rate of product formation at 25°C was slightly higher with the lower substrate concentration (3 M mannose) , but as expected, the yield was higher with the higher substrate concentration (7 M) (14% as compared with 12%) . However, whereas the maximum product yield was obtained after ca five days with 3 M mannose, more than ten days were required with 7 M initial concentration of mannose. Shorter reaction times can be obtained with more enzyme and/or a higher reaction temperature. The mannosylation of the threonine derivative was less efficient and seemed to be disturbed by partial inactivation of the α-mannosidase, as indicated by that the yield increased from 5 to 8% (HPLC) by using higher enzyme concentration (25 units per ml instead of 10 units per ml) . Further variations and modifications of the foregoing will be apparent to those skilled in the art and such variations and modifications are attended to be encompassed by the claims that are appended hereto. Swedish Priority Application 9304316-4, filed on December 24, 1993, is relied on and incorporated by reference.

Claims: CLAIMS 1. Method for synthesis of O-glycosylated amino acid or peptide or derivatives thereof, characterized by the use of a glycosyl donor which is a monosaccharide in pyranose or furanose form or which is an oligosaccharide, and by the use of an acceptor which is a hydroxyl group containing amino acid or peptide derivative which has been modified in its N-terminal α- amino group and in which the C-terminal carboxyl group is non- modified, employing an exo- or an endoglycosidase (EC 3.2) as catalyst in an equilibrium reaction and that the product is isolated from the reaction mixture or is used directly or after partial or complete isolation in a continued synthesis with chemical methods or with enzymatic methods. 2. Method for synthesis of glycosylated amino acid or peptide and derivatives of any of these according to claim 1 above, characterized in that the glycosyl donor consists of or contains at least one amino sugar, as for example N-acetyl-D- glucosamine, N-acetyl-D-galactosamine, N-acetyl-D-mannosamine or consists of or contains at least one L-fucose unit. 3. Method according to claims 1 or 2 above characterized in that the donor exists in high concentration in the reaction mixture. 4. A method for the synthesis of O-glycosylated amino acid or peptide or derivative thereof, said method comprising reacting (a) a glycosyl donor which is a monosaccharide in pyranose or furanose form or which is an oligosaccharide, (b) an acceptor which is a hydroxyl group containing amino acid or peptide derivative which has been modified in its N-terminal c*- amino group and in which the C-terminal carboxyl group is non- modified, and (c) an enzyme which is an EC Group 3.2 exo- or an endoglycosidase in an equilibrium reaction. 5. The method according to claim 4, further comprising isolating the product from the reaction mixture. 6. The method according to claim 4, wherein said glycosyl donor contains at least one amino sugar. 7. The method according to claim 6, wherein said glycosyl donor is N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, or N- acety1-D-mannosamine. 8. The method according to claim 4, wherein said glycosyl donor contains at least one L-fucose unit. 9. The method according to claim 4, wherein said glycosyl donor is present in high concentration in the reaction mixture. 10. The method according to claim 9, wherein said glycosyl donor is present in an amount greater than 1 M. 11. The method according to claim 4, wherein said modified N-terminal α-amino group contains an amino protection group or an amino acid or peptide group which is protected in the α-N- terminal group with an amino protection group. 12. The method according to claim 11, wherein said amino protection group is an alkoxy or aliphatic protection group. 13. The method according to claim 12, wherein said alkoxy or aliphatic protection group is acetyl, benzyloxycarbonyl-, allyloxycarbonyl-, t-butyloxycarbonyl-, formyl-, acetyl-, fenacetyl-, 4-metoxybenzyloxycarbonyl-, or 9-fluorenyl methyloxycarbonyl. 14. An O-glycosylated amino acid or peptide or derivative thereof produced by the method according to claim 4. 15. A method of using an acceptor which is a hydroxyl group containing amino acid or peptide derivative which has been modified in its N-terminal α-amino group and in which the C- terminal carboxyl group is non-modified to produce an O- glycosylated amino acid or peptide or derivative thereof, said method comprising reacting said acceptor with a glycosyl donor which is a monosaccharide in pyranose or furanose form or which is an oligosaccharide and an exo- or an endoglycosidase (EC 3.2) in an equilibrium reaction to produce said O-glycosylated amino acid or peptide or derivatives thereof. 16. Use of the O-glycosylated amino acid or peptide or derivatives according to claim 1. 17. Use of the O-glycosylated amino acid or peptide or derivatives according to claim 1 for the synthesis of O- glycosylated amino acid or peptide or derivatives of Gal/51- 3GlcNAc or Gal/ l-3GalNAc by the use of /5-D-galactosidase as catalyst, nitrophenyl β-D-galactoside or lactose as donor and GlcNAcjS-(N-X)Y or GalNAcα-(N-X)Y as acceptor. 18. Use of the O-glycosylated amino acid or peptide or derivatives according to claim 1 for the synthesis of O- glycosylated amino acid or peptide or derivatives of Gal/31- 4GlcNAc/3-(N-X)Y by the use of β-galactosidase as catalyst, nitrophenyl / 5-D-galactoside or lactose as donor and GlcNAc/3-(N- X)Y as acceptor. 19. Use of the O-glycosylated amino acid or peptide or derivatives according to claim 1 for the synthesis of 0- glycosylated amino acid or peptide or derivatives of Manαl-2Manα by the use of α-D-mannosidase as catalyst, nitrophenyl α-D- mannopyranoside as donor and Manα-(N-X)Y as acceptor.

Inventors: NILSSON KURT, NILSSON, KURT
Applicants: NILSSON KURT, NILSSON, KURT
Application year: 1994
Main classification: C12P19
IPC codes: C12P19, C12P21